Stimulated emission depletion (STED) fluorescence light microscopy allows for a spatial resolution in imaging a structure which is marked with fluorescent entities in a sample, or in delimiting the observation volume in FCS, which surpasses the diffraction barrier. This is achieved in that, after exciting the fluorescent entities for fluorescence by the excitation light, the spatial distribution of the excited fluorescent entities is altered by de-excitation or STED light stimulating the excited fluorescent entities for stimulated emission. Whereas the excitation light may only be focussed to a focal area with spatial dimensions above the diffraction barrier, a zero point of the intensity of the de-excitation light in which the excited fluorescent entities are not kept dark in that they are returned to their ground state by stimulated emission and are thus still able to emit fluorescence light, and around which the fluorescent entities are completely de-excited again, may be made much smaller. For example, the zero point of the intensity distribution of the de-excitation light may be defined by destructive interference of different components of the de-excitation light; and with increasing overall intensity of the de-excitation light the boundary of such a zero point beyond which the fluorescent entities are completely de-excited is closely drawn around a geometric point.
In STED fluorescence light microscopy, the excitation light is usually applied to the sample in pulses. Typically, the de-excitation light is also applied to the sample in pulses, and a detector for registering the fluorescence light emitted out of the zero point of the intensity distribution of the de-excitation light is only turned on directly after each pulse of the de-excitation light fades out.
As already indicated above, however, the de-excitation light needs to have a rather high intensity to yield a high spatial resolution. Pulsed lasers delivering high intensity pulses are expensive, particularly, if the pulses have to be very short, i.e. shorter than the lifetime of the excited state of the fluorescent entities, during which the excited state already decays by spontaneous emission of fluorescence light, as otherwise there would be no excited fluorescent entities left after each pulse of the de-excitation light, even in the zero point of its intensity distribution.
Besides applying both the excitation light and the de-excitation light in pulses as explained above, U.S. Pat. No. 5,731,588 also discloses to use a continuous wave laser as an excitation light source to save cost. Even then, the detector registering the fluorescence light spontaneously emitted out of the zero point of the intensity distribution of the de-excitation light is only turned on directly after each pulse of the de-excitation light has passed the sample.
US 2010/0176307 A1 discloses STED fluorescence light microscopy with two-photon excitation in which excitation light is applied to a sample in pulses at such a wavelength that fluorescent entities in the sample are excited for the emission of fluorescence light in a multi-photon process. The de-excitation or STED light is applied to the sample as a continuous wave, and the fluorescence light spontaneously emitted by the excited fluorescent entities in the sample is continuously recorded over several pulses of the excitation light. Due to the multi-photon process used for exciting the fluorescent entities in the sample, the spatial distribution of the excited fluorescent entities is assumed to not extend far beyond the zero point of the intensity distribution of the de-excitation light. Thus, it becomes possible to considerably save cost in that the de-excitation light is applied to the sample by a continuous wave laser, and in that the fluorescence light spontaneously emitted by the sample is continuously registered. In registering the spontaneously emitted fluorescence light, the excitation light, the de-excitation light and the stimulated emission from the fluorescent entities are blocked by a suitable edge filter or narrow-band bandpass filter and/or by means of a polarization filter, if the excitation light and the de-excitation light are suitably polarized.
The signal yield in any fluorescence light microscopic method using multi-photon excitation, however, is only small, and suitable pulsed light sources for exciting fluorescent entities in a multi-photon process, which have a suitable output intensity, are expensive.
A method of STED fluorescence light microscopy called CW-STED has been published which differs from that one disclosed in US 2010/0176307 A1 in that the excitation light applied to the sample in pulses excites the fluorescent entities for fluorescence in a simple one-photon process.
Both the two-photon excitation method disclosed in US 2010/0176307 A1 and CW-STED have been found to lack something of the expected spatial resolution. In fact, the images of a known structure marked with fluorescent entities look somewhat blurred as compared to images of the same structure obtained by pulsed excitation light and pulsed de-excitation light and by registering the spontaneously emitted fluorescence light only after each pulse of the de-excitation light.
Thus, a need remains for STED fluorescence light microscopy using a low-cost continuous wave laser but nevertheless exhibiting an uncompromised spatial resolution.